Most end-stage cells in renewing systems are short-lived and must be replaced continuously throughout life. For example, blood cells originate from a self-renewing population of multipotent hematopoietic stem cells (HSC). Hematopoietic stem cells are a subpopulation of hematopoietic cells. Hematopoietic cells can be obtained, for example, from bone marrow, umbilical cord blood or peripheral blood (either unmobilized or mobilized with an agent such as G-CSF), hematopoietic cells include the stem cell population, progenitor cells, differentiated cells, accessory cells, stromal cells and other cells that contribute to the environment necessary for production of mature blood cells. Hematopoietic progenitor cells are a subset of stem cells which are more restricted in their developmental potency. Progenitor cells are able to differentiate into only one or two lineages (e.g., BFU-E and CFU-E which give rise only to erythrocytes or CFU-GM which give rise to granulocytes and macrophages) while stem cells (such as CFU-MIX or CFU-GEMM) can generate multiple lineages and/or other stem cells. Because the hematopoietic stem cells are necessary for the development of all of the mature cells of the hematopoietic and immune systems, their survival is essential in order to reestablish a fully functional host defense system in subjects treated with chemotherapy or other agents.
Hematopoietic cell production is regulated by a series of factors that stimulate growth and differentiation of hematopoietic cells, some of which, for example erythropoietin, GM-CSF and G-CSF, are currently used in clinical practice. One part of the control network which has not been extensively characterized, however, is the physiological mechanism that controls the cycling status of stem cells (Eaves et al. Blood 78:110–117, 1991; Lord, in Stem Cells (C. S. Potten, Ed.) pp 401–22, 1997 (Academic Press, NY)).
Early studies by Lord and coworkers showed the existence of soluble protein factors in normal and regenerating bone marrow extracts which could either inhibit or stimulate stem cell proliferation (reviewed in: Lord and Wright, Blood Cells 6:581–593, 1980; Wright and Lorimore, Cell Tissue Kinet. 20:191–203, 1987; Marshall and Lord, Int Rev. Cyt. 167:185–261, 1996). These activities were designated stem cell inhibitor (SCI) and stem cell stimulator (SCS), respectively.
To date, no candidate SCS molecules have been purified from bone marrow extracts prepared as described by Lord et al. (reviews referenced above). Purification of either SCS or SCI from primary sources was not accomplished due to the difficulties inherent in an in vivo assay requiring large numbers of irradiated mice. In an attempt to overcome these problems Pragnell and co-workers developed an in vitro assay for primitive hematopoietic cells (CFU-A) and screened cell lines as a source of the inhibitory activity (see Graham et al. Nature 344:442–444, 1990). As earlier studies had identified macrophages as possible sources for SCI (Lord et al. Blood Cells 6:581–593, 1980), a mouse macrophage cell line, J774.2, was selected (Graham et al. Nature 344:442–444, 1990). The conditioned medium from this cell line was used by Graham et al. for purification; an inhibitory peptide was isolated which proved to be identical to the previously described cytokine macrophage inflammatory protein 1-alpha (MIP-1α). Receptors for MIP-1α have been cloned; like other chemokine receptors, these MIP-1α receptors are seven-transmembrane domain (or “G-linked”) receptors which are coupled to guanine nucleotide (GTP) binding proteins of the Ginhibitory subclass (“Gi”) (reviewed in Murphy, Cytokine & Growth Factor Rev. 7:47–64, 1996). The “inhibitory” designation for the Gi subclass refers to its inhibitory activity on adenylate cyclase.
MIP-1α was isolated from a cell line, not from primary material. While Graham et al. observed that antibody to MIP-1α abrogated the activity of a crude bone marrow extract, other workers have shown that other inhibitory activities are important. For example, Graham et al. (J. Exp. Med. 178:925–32, 1993) have suggested that TGFβ. not MIP-1α, is a primary inhibitor of hematopoietic stem cells. Further, Eaves et al. (PNAS 90:12015–19, 1993) have suggested that both MIP-1α and TGFβ are present at sub optimal levels in normal bone marrow and that inhibition requires a synergy between the two factors.
Recently, mice have been generated in which the MIP-1α gene has been deleted by homologous recombination (Cook et al., Science 269:1583–5, 1995). Such mice have no obvious derangement of their hematopoietic system, calling into question the role of MIP-1α as a physiological regulator of stem cell cycling under normal homeostatic conditions. Similarly, although transforming growth factor beta (TGFβ) also has stem cell inhibitory activities, the long period of time it takes for stem cells to respond to this cytokine suggests that it is not the endogenous factor present in bone marrow extracts; further, neutralizing antibodies to TGFβ do not abolish SCI activity in bone marrow supernatants (Hampson et al., Exp. Hemat. 19:245–249, 1991).
Other workers have described additional stem cell inhibitory factors. Frindel and coworkers have isolated a tetrapeptide from fetal calf marrow and from liver extracts which has stem cell inhibitory activities (Lenfant et al., PNAS 86:779–782, 1989). Paukovits et al. (Cancer Res. 50:328–332, 1990) have characterized a pentapeptide which, in its monomeric form, is an inhibitor and, in its dimeric form, is a stimulator of stem cell cycling. Other factors have also been claimed to be inhibitory in various in vitro systems (see Wright and Pragnell in Bailliere's Clinical Haematology v. 5, pp. 723–39, 1992 (Bailliere Tinadall, Paris); Marshall and Lord, Int Rev. Cyt. 167:185–261, 1996).
Tsyrlova et al., SU 1561261 A1, disclosed a purification process for a stem cell proliferation inhibitor.
Commonly owned applications WO 94/22915 and WO96/10634 disclose an inhibitor of stem cell proliferation, and are hereby incorporated by reference in their entirety.
To date, none of these factors have been approved for clinical use. However, the need exists for effective stem cell inhibitors. The major toxicity associated with chemotherapy or radiation treatment is the destruction of normal proliferating cells which can result in bone marrow suppression or gastrointestinal toxicity. An effective stem cell inhibitor will protect these cells and allow for the optimization of these therapeutic regimens. Just as there is a proven need for a variety of stimulatory cytokines (i.e., cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-1, IL-13, IL-14, IL-15, G-CSF, GM-CSF, erythropoietin, thrombopoietin, stem cell factor, flk2/flt3 ligand, etc., which stimulate the cycling of hematopoietic cells) depending upon the clinical situation, so too it is likely that a variety of inhibitory factors will be needed to address divergent clinical needs.
Further, there is a need to rapidly reverse the activity of such an inhibitor. The original studies of Lord et al. (reviews referenced above) demonstrated that the inhibitory activity could be reversed by addition of the stimulatory activity. While a variety of stem cell stimulatory cytokines has been identified (see above), none has been demonstrated to represent the activity described by Lord and coworkers as being present in bone marrow extracts and of being able to reverse the activity of the inhibitor.
Hematopoietic progenitors and stem cells primarily reside in the bone marrow in normal adults. Under certain conditions, for example chemotherapy or treatment with cytokines such as G-CSF, large numbers of progenitors and stem cells egress into the peripheral blood, a process referred to as “mobilization” (reviewed in Simmons et al., Stem Cells 12 (suppl 1): 187–202, 1994; Scheding et al. Stem Cells 12 (suppl 1):203–11, 1994; Mangan, Sem. Oncology 22:202–9, 1995; Moolten, Sem. Oncology 22:271–90, 1995). Recent published data suggest that the vast majority of mobilized progenitors are not actively in cell cycle (Roberts and Metcalf, Blood 86:1600-, 1995; Donahue et al., Blood 87:1644-, 1996; Siegert and Serke, Bone Marrow Trans. 17:467- 1996; Uchida et al., Blood 89:465–72, 1997).
Hemoglobin is a highly conserved tetrameric protein with molecular weight of approximately 64,000 Daltons. It consists of two alpha and two beta chains. Each chain binds a single molecule of heme (ferroprotoporphyrin IX), an iron-containing prosthetic group. Vertebrate alpha and beta chains were probably derived from a single ancestral gene which duplicated and then diverged; the two chains retain a large degree of sequence identity both between themselves and between various vertebrates (see FIG. 16A). In humans, the alpha chain cluster on chromosome 16 contains two alpha genes (alpha1 and alpha2) which code for identical polypeptides, as well as genes coding for other alpha-like chains: zeta, theta and several non-transcribed pseudogenes (see FIG. 16B for cDNA and amino acid sequences of human alpha chain). The beta chain cluster on chromosome 11 consists of one beta chain gene and several beta-like genes: delta, epsilon, G gamma and A gamma, as well as at least two unexpressed pseudogenes (see FIG. 16C for cDNA and amino acid sequences of human beta chain).
The expression of these genes varies during development. In human hematopoiesis, which has been extensively characterized, embryonic erythroblasts successively synthesize tetramers of two zeta chains and two epsilon chains (Gower I), two alpha chains and two epsilon chains (Gower II) or two zeta chains and two gamma chains (Hb Portland). As embryogenesis proceeds, the predominant form consists of fetal hemoglobin (Hb F) which is composed of two alpha chains and two gamma chains. Adult hemoglobin (two alpha and two beta chains) begins to be synthesized during the fetal period; at birth approximately 50% of hemoglobin is of the adult form and the transition is complete by about 6 months of age. The vast majority of hemoglobin (approximately 97%) in the adult is of the two alpha and two beta chain variety (Hb A) with small amounts of Hb F or of delta chain (Hb A2) being detectable.
Several methods have been used to express recombinant hemoglobin chains in E. coli and in yeast (e.g., Jessen et al., Methods Enz. 231:347–364, 1994; Looker et al. Methods Enz. 231:364–374, 1994; Ogden et al., Methods Enz. 231:374–390, 1994; Martin de Llano et al., Methods Enz. 231:364–374, 1994). It has thus far not been possible to express isolated human alpha chain in high yields by recombinant methods (e.g., Hoffman et al., PNAS 87:8521–25, 1990; Heman et al., Biochem. 31:8619–28, 1992). Apparently, the isolated alpha chain does not assume a stable conformation and is rapidly degraded in E. coli. Co-expression of beta chain with alpha chain results in increased expression of both (Hoffman et al. and Hernan et al., op. cit.). While the alpha chain has been expressed as a fusion protein with a portion of the beta chain and a factor Xa recognition site (Nagai and Thorgersen, Methods Enz. 231:347–364, 1994) it is expressed as an insoluble inclusion body under these conditions.
Both the beta chain and the alpha chain contain binding sites for haptoglobin. Haptoglobin is a serum protein with extremely high affinity for hemoglobin (e.g., Putnam in The Plasma Proteins—Structure, Function and Genetic Control (F. W. Putnam, Ed.) Vol. 2, pp 1–49 (Academic Press, NY); Hwang and Greer, JBC 255:3038–3041, 1980). Haptoglobin transport to the liver is the major catabolic pathway for circulating hemoglobin. There is a single binding site for haptoglobin on the alpha chain (amino acids 121–127) and two on the beta chain (amino acid regions 11–25 and 131–146) (Kazim and Atassi, Biochem J. 197:507–510, 1981; McCormick and Atassi, J. Prot. Chem. 9:735–742, 1990).
Biologically active peptides with opiate activity have been obtained by proteolytic degradation of hemoglobin (reviewed in Karelin et al., Peptides 16:693–697, 1995). Hemoglobin alpha chain has an acid-labile cleavage site between amino acids 94–95 (Shaeffer, J. Biol. Chem. 269:29530–29536, 1994).
Kregler et al. (Exp. Hemat. 9:11–21, 1981) have disclosed that hemoglobin has an enhancing activity on mouse bone marrow CFU-C progenitor colonies. Such assays demonstrate effects on CFU-GM and CFU-M progenitor populations as opposed to stem cells such as CFU-MIX. The authors observed activity in both isolated alpha and beta chains of hemoglobin. This activity was abolished by treatment with N-ethylmaleimide, which suggested to Kregler et al. that sulfhydryl groups were necessary. This observation, coupled with the fact that the stimulatory activity was resistant to trypsin digestion, suggested to Kregler et al. that the C-terminal hydrophobic domain or “core” region was responsible for the activity. Moqattash et al. (Acta. Haematol. 92:182–186, 1994) have disclosed that recombinant hemoglobin has a stimulatory effect on CFU-E, BFU-E and CFU-GM progenitor cell number which is similar to that observed with hemin. Mueller et al. (Blood 86:1974, 1995) have disclosed that purified adult hemoglobin stimulates erythroid progenitors in a manner similar to that of hemin.
Petrov et al. (Bioscience Reports 15:1–14, 1995) disclosed the use of a “nonidentified myelopeptide mixture” in the treatment of congenital anemia in the Wv/Wv mouse. The mixture increased the number of spleen colonies, especially those of the erythroid type.
Heme and hemin have been extensively examined with regard to their influences on hematopoiesis (see S. Sassa, Seminars Hemat. 25:312–20, 1988 and N. Abraham et al., Int. J. Cell Cloning 9:185–210, 1991 for reviews). Heme is required for the maturation of erythroblasts; in vitro, hemin (chloroferroprotoporphyrin IX—i.e., heme with an additional chloride ion) increases the proliferation of CFU-GEMM, BFU-E and CFU-E. Similarly, hemin increases cellularity in long-term bone marrow cultures.
“Opiates” are substances with analgesic properties similar to morphine, the major active substance in opium. Opiates can be small organic molecules, such as morphine and other alkaloids or synthetic compounds, or endogenous peptides such as enkephalins, endorphins, dynorphins and their synthetic derivatives. Endogenous opiate peptides are produced in vivo from larger precursors—pre-proenkephalin A for Met- and Leu-enkephalins, pre-proopiomelanocortin for α, β, and γ endorphins, and pre-prodynorphin for dynorphins A and B, α-neoendorphin and β-neoendorphin. In addition, peptides with opiate activity can be obtained from non-classical sources such as proteolysis or hydrolysis of proteins such as α or β casein, wheat gluten, lactalbumin, cytochromes or hemoglobin, or from other species such as frog skin (dermorphins) or bovine adrenal medulla. Such peptides have been termed “exorphins” in contrast to the classical endorphins; they are also referred to as a typical opiate peptides (Zioudrou et al., JBC 254:2446–9, 1979; Quirion and Weiss, Peptides 4:445, 1983; Loukas et al. Biochem. 22:4567, 1983; Brantl, Eur. J. Pharm. 106:213–14, 1984; Brantl et al., Eur. J. Pharm. 111:293–4, 1985; Brantl et al., Eur. J. Pharm. 125:309–10, 1986; Brantl and Neubert, TIPS 7:6–7,1986; Glamsta et al., BBRC 184:1060–6, 1992; Teschemacher, Handbook Exp. Pharm. 104:499–28, 1993; Petrov et al., Bioscience Reports 15:1–14, 1995; Karelin et al., Peptides 16:693–7, 1995). Other endogenous peptides, such as the Tyr-MIF-1 family, have also been shown to have opiate activity (Reed et al., Neurosci. and Biobehav. Rev. 18:519–25, 1994).
Opiates exert their actions by binding to three main pharmacological classes of endogenous opiate receptors—mu, delta, and kappa. Receptors representing each pharmacological class have been cloned and shown to be G-linked receptors coupled to Gi (reviewed in: Reisine and Bell, TINS 16: 506–510, 1993; Uhl et al., TINS 17:89–93, 1994; Knapp et al., FASEB J. 9:516–525, 1995; Satoh and Minami, Pharm. Ther. 68:343–64, 1995; Kieffer, Cell. Mol. Neurobiol. 15:615–635, 1995; Reisine, Neuropharm. 34:463–472, 1995; Zaki et al., Ann. Rev. Pharm. Toxicol., 36:379–401, 1996).
Specific agonists and antagonists are available for each receptor type—e.g., for mu receptors (which are selectively activated by DAIMGO and DALDA and selectively antagonized by CTOP and naloxonazine), for kappa receptors (which are selectively activated by GR 89696 fumarate or U-69593 and selectively antagonized by nor—binaltorphimine hydrochloride) and for delta receptors (which are selectively activated by DADLE and DPDPE and selectively antagonized by natrindole). In addition, there are broad-spectrum antagonists (such as naloxone) and agonists (such as etorphine) which act on all three receptor subtypes.
Both classical and a typical opiate peptides can be chemically altered or derivatized to change their specific opiate receptor binding properties (reviewed in Hruby and Gehrig, Med. Res. Rev. 9:343–401, 1989; Schiller, Prog. Med. Chem. 28: 301–40, 1991; Teschemacher, Handbook Exp. Pharm. 104:499–28, 1993; Handbook of Experimental Pharmacology, A. Hertz (Ed.) volumes 104/II and 104/II, 1993, Springer Verlag, Berlin; Karelin et al., Peptides 16:693–7, 1995). Examples include derivatives of dermorphin (e.g., DALDA) and enkephalins (e.g., DADLE, DAIMGO or DAMME). Peptides which do not normally bind to opiate receptors, such as somatostatin, can also be derivatized to exhibit specific opiate receptor binding (e.g., CTOP (Hawkins et al., J. Pharm. Exp. Ther. 248:73, 1989)). Analogs can also be derived from alkaloids such as morphine with altered receptor binding properties (e.g., heroin, codeine, hydromorphone, oxymorphone, levorphanol, levallorphan, codeine, hydrocodone, oxycodone, nalorphine, naloxone, naltrexone, buprenorphine, butanorphanol and nalbuphine); in addition, small molecules structurally unrelated to morphine can also act on opiate receptors (e.g., meperidine and its congeners alphaprodine, diphenoxylate and fentanyl) (see Handbook of Experimental Pharmacology, op. cit.; Goodman and Gilman's The Pharmacological Basis of Therapeutics. 7th Ed., A. G. Gilman, L. S. Goodman, T. W. Rall and F. Murad (Eds.) 1985 Macmillan Publishing Co. NY).
The endogenous opiate peptides (enkephalins, endorphins and dynorphins) have a conserved N-terminal tetrapeptide Tyr-Gly-Gly-Phe, followed by Leu or Met and any remaining C-terminal sequence. Removal of the hydroxyl group on the N-terminal Tyr (resulting in an N-terminal Phe) results in a dramatic loss of activity for Met-enkephalin. These structural data led to the “message-address” hypothesis whereby the N-terminal “message” confers biological activity while the C-terminal “address” confers specificity and enhanced potency (Chavkin and Goldstein, PNAS 78:6543–7, 1981). Exorphins generally have a Tyr-Pro replacing the N-terminal Tyr-Gly of classical opiate peptides; the proline residue is thought to confer higher stability against aminopeptidase degradation (Shipp et al., PNAS 86: 287-, 1989; Glamsta et al., BBRC 184:1060–6, 1992).
Recently an orphan receptor (“ORL1”) was cloned by virtue of sequence relatedness to the mu, delta and kappa opiate receptors (Mollereau et al., FEBS 341:33–38, 1994; Fukuda et al., FEBS 343:42–46, 1994; Bunzow et al., FEBS 347:284–8, 1994; Chen et al., FEBS 347:279–83, 1994; Wang et al., FEBS 348:75–79, 1994; Keith et al., Reg. Peptides 54 143–4, 1994; Wick et al., Mol. Brain Res. 27: 37–44, 1994, Halford et al., J. Neuroimmun. 59:91–101, 1995). The ligand for this receptor, variously called nociceptin or orphanin FQ (referred to hereafter as “nociceptin”) has been cloned and shown to be a heptadecapeptide which is derived from a larger precursor (Meunier et al., Nature 377:532–535, 1995; Reinscheid et al., Science 270:792–794, 1995). It was demonstrated to have pronociceptive, hyperalgesic functions in vivo, as opposed to classical opiates which have analgesic properties. Nociceptin has a Phe-Gly-Gly-Phe . . . N-terminal motif in contrast to the Tyr-Gly-Gly-Phe . . . N-terminal motif of classical opiate peptides discussed above. In keeping with the requirement for an N-terminal Tyr for opiate activity in classical opiate peptides, nociceptin exhibits little or no affinity for the mu, kappa or delta opiate receptors. Similarly, the broad-spectrum opiate antagonist naloxone has no appreciable affinity for ORL1.
Enkephalins have been observed to have effects on murine hematopoiesis in vivo under conditions of immobilization stress (Goldberg et al., Folia Biol. (Praha) 36:319–331, 1990). Leu-enkephalin inhibited and met-enkephalin stimulated bone marrow hematopoiesis. These effects were indirect, Goldberg et al. believed due to effects on glucocorticoid levels and T lymphocyte migration. Krizanac-Bengez et al. (Biomed. & Pharmacother. 46:367–43, 1992; Biomed. & Pharmacother. 49:27–31, 1995; Biomed. & Pharmacother. 50:85–91, 1996) looked at the effects of these compounds in vitro. Pre-treatment of murine bone marrow with Met- or Leu-enkephalin or naloxone affected the number of GM progenitor cells observed in a colony assay. This effect was highly variable and resulted in suppression, stimulation or no effect; further, there was no clear dose-response. This variability was ascribed by Krizanac-Bengez et al. to circadian rhythms and to accessory cells.
Recently, it has been demonstrated that mice in which the mu opiate receptor has been deleted by homologous recombination have elevated numbers of CFU-GM, BFU-E and CFU-GEMM per femur. Marrow and splenic progenitors were more rapidly cycling in these mu receptor knockout mice compared to normal mice. It was not determined if these effects were due to a direct or indirect effect on bone marrow stem cells resulting from the absence of the mu receptor in these animals (Broxmeyer et al., Blood 88:338a, 1997).
I. Chemotherapy and Radiotherapy of Cancer
Productive research on stimulatory growth factors has resulted in the clinical use of a number of these factors (erythropoietin, G-CSF, GM-CSF, etc.). These factors have reduced the mortality and morbidity associated with chemotherapeutic and radiation treatments. Further clinical benefits to patients who are undergoing chemotherapy or radiation could be realized by an alternative strategy of blocking entrance of stem cells into cell cycle thereby protecting them from toxic side effects. The reversal of this protection will allow for rapid recovery of bone marrow function subsequent to chemo- or radiotherapy.
II. Bone Marrow and Stem Cell Transplantation, Ex Vivo Stem Cell Expansion and Tumor Purging
Bone marrow transplantation (BMT) is a useful treatment for a variety of hematological, autoimmune and malignant diseases. Current therapies include hematopoietic cells obtained from umbilical cord blood, fetal liver or from peripheral blood (either unmobilized or mobilized with agents such as G-CSF or cyclophosphamide) as well as from bone marrow; the stem cells may be unpurified, partially purified (e.g., affinity purification of the CD34+ population) or highly purified (e.g., through fluorescent activated cell sorting using markers such as CD34, CD38 or rhodamine). Ex vivo manipulation of hematopoietic cells is currently being used to expand primitive stem cells to a population suitable for transplantation. Optimization of this procedure requires: (1) sufficient numbers of stem cells able to maintain long term reconstitution of hematopoiesis; (2) the depletion of graft versus host-inducing T-lymphocytes and (3) the absence of residual malignant cells. This procedure can be optimized by including a stem cell inhibitor(s) and/or a stem cell stimulator(s).
The effectiveness of purging of hematopoietic cells with cytotoxic drugs in order to eliminate residual malignant cells is limited due to the toxicity of these compounds for normal hematopoietic cells and especially stem cells. There is a need for effective protection of normal cells during purging; protection can be afforded by taking stem cells out of cycle with an effective inhibitor.
III. Peripheral Stem Cell Harvesting
Peripheral blood stem cells (PBSC) offer a number of potential advantages over bone marrow for autologous transplantation. Patients without suitable marrow harvest sites due to tumor involvement or previous radiotherapy can still undergo PBSC collections. The use of blood stem cells eliminates the need for general anesthesia and a surgical procedure in patients who would not tolerate this well. The apheresis technology necessary to collect blood cells is efficient and widely available at most major medical centers. The major limitations of the method are both the low normal steady state frequency of stem cells in peripheral blood and their high cycle status after mobilization procedures with drugs or growth factors (e.g., cyclophosphamide, G-CSF, stem cell factor). An effective stem cell inhibitor will be useful to return such cells to a quiescent state, thereby preventing their loss through differentiation.
IV. Treatment of Hyperproliferative Disorders
A number of diseases are characterized by a hyperproliferative state in which dysregulated stem cells give rise to an overproduction of end stage cells. Such disease states include, but are not restricted to, psoriasis, in which there is an overproduction of epidermal cells, premalignant conditions in the gastrointestinal tract characterized by the appearance of intestinal polyps, and acquired immune deficiency syndrome (AIDS) where early stem cells are not infected by HIV but cycle rapidly resulting in stem cell exhaustion. A stem cell inhibitor will be useful in the treatment of such conditions.
V. Treatment of Hypoproliferative Disorders
A number of diseases are characterized by a hypoproliferative state in which dysregulated stem cells give rise to an underproduction of end stage cells. Such disease states include myelodysplatic syndromes or aplastic anemia, in which there is an underproduction of blood cells, and conditions associated with aging where there is a deficiency in cellular regeneration and replacement A stem cell stimulator will be useful in the treatment of such conditions.
VI. Gene Transfer
The ability to transfer genetic information into hematopoietic cells is currently being utilized in clinical settings. Hematopoietic cells are a useful target for gene therapy because of ease of access, extensive experience in manipulating and treating this tissue ex vivo and because of the ability of blood cells to permeate tissues. Furthermore, the correction of certain human genetic defects can be possible by the insertion of a functional gene into the primitive stem cells of the human hematopoietic system.
There are several limitations for the introduction of genes into human hematopoietic cells using either retrovirus vectors or physical techniques of gene transfer: (1) The low frequency of stem cells in hematopoietic tissues has necessitated the development of high efficiency gene transfer techniques; and (2) more rapidly cycling stem cells proved to be more susceptible to vector infection, but the increase of the infection frequency by stimulation of stem cell proliferation with growth factors produces negative effects on long term gene expression, because cells containing the transgenes are forced to differentiate irreversibly and lose their self-renewal. These problems can be ameliorated by the use of a stem cell inhibitor to prevent differentiation and loss of self-renewal and a stem cell stimulator to regulate the entry of stem cells into cycle and thereby facilitate retroviral-mediated gene transfer.